As a result of recent innovations in drug discovery, including genomics, combinatorial chemistry and high throughput screening, the number of drug candidates available for clinical testing exceeds the pharmaceutical industry's development and economic capacity. In 1998, the world's top pharmaceutical and biotechnology companies spent more than $50 billion on research and development, more than one-third of which was spent directly on clinical development. As the result of a number of factors, including increased competition and pressure from managed care organizations and other payors, the pharmaceutical industry is seeking to increase the quality, including the safety and efficacy of new drugs brought to market, and to improve the efficiency of clinical development.
Recent drug discovery innovations, therefore, have contributed to a clinical trials bottleneck. The numbers of therapeutic targets being identified and lead compounds being generated far exceed the capacity of pharmaceutical companies to conduct clinical trials as they are currently performed. Further, as the industry currently estimates that the average cost of developing a new drug is approximately $500 million, it is prohibitively expensive to develop all of the potential drug candidates.
The pharmaceutical industry is being forced to seek equivalent technological improvements in drug development. Clinical trials remain very expensive and very risky, and often decision making is based on highly subjective analyses. As a result, it is often difficult to determine the patient population for whom a drug is most effective, the appropriate dose for a given drug and the potential for side effects associated with its use. Not only does this lead to more failures in clinical development, it can also lead to approved products that may be inappropriately dosed, prescribed, or cause dangerous side effects. With an increasing number of drugs in their pipelines, pharmaceutical companies require technologies to identify objective measurements of a drug candidate's safety and efficacy profile earlier in the drug development process.
Biological markers are characteristics that when measured or evaluated have a discrete relationship or correlation as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention. Pharmacologic responses to therapeutic intervention include, but are not limited to, response to the intervention generally (e.g., efficacy), dose response to the intervention, side effect profiles of the intervention, and pharmacokinetic properties such as the rate of drug metabolism and the identity of the drug metabolites. Response may be correlated with either efficacious or adverse (e.g., toxic) changes. Biological markers include patterns of cells or molecules that change in association with a pathological process and have diagnostic and/or prognostic value. Biological markers may include levels of cell populations and their associated molecules, levels of soluble factors, levels of other molecules, gene expression levels, genetic mutations, and clinical parameters that can be correlated with the presence and/or progression of disease. In contrast to such clinical endpoints as disease progression or recurrence or quality of life measures (which typically take a long time to assess), biological markers may provide a more rapid and quantitative measurement of a drug's clinical profile. Single biological markers currently used in both clinical practice and drug development include cholesterol, prostate specific antigen (“PSA”), CD4 T cells and viral RNA. Unlike the well known correlations between high cholesterol and heart disease, PSA and prostate cancer, and decreased CD4 positive T cells and viral RNA in AIDS, the biological markers correlated with most other diseases have yet to be identified. As a result, although both government agencies and pharmaceutical companies are increasingly seeking development of biological markers for use in clinical trials, the use of biological markers in drug development has been limited to date.
There is a need for a biological marker identification system that is capable of sorting through the vast amounts of information needed to establish the correlation of the biological markers with disease, disease progression and response to therapy. Such a biological marker identification system is described in U.S. Provisional Patent Application Serial No. 60/131,105, entitled “Biological Marker Identification System”, filed 26 Apr. 1999, and in the commonly-owned United States Utility Application filed concurrently with this application, entitled “Phenotype and Biological Marker Identification System,” both of which are specifically incorporated herein by reference in its entirety. This technology includes the instrumentation and assays required to measure hundreds to thousands of biological markers, an informatics system to allow this data to be easily accessed, software to correlate the patterns of markers with clinical data and the ability to utilize the resulting information in the drug development process. The system extensively utilizes Microvolume Laser Scanning Cytometry (MLSC).
In preferred embodiments of the marker identification system, a biological fluid is contacted with one or more fluorescently-labeled detection molecules that can bind to specific molecules in that fluid. Typically, the biological fluid is a blood sample, and the detection molecule is a fluorescent dye-labeled antibody specific for a cell-associated molecule that is present on, or within, one or more sub-types of blood cell. The labeled sample is then placed in a capillary tube, and the tube is mounted on a MLSC instrument. This instrument scans laser light through a microscope objective onto the blood sample. Fluorescent light emitted from the sample is collected by the microscope objective and passed to a series of photomultipliers where images of the sample in each fluorescent channel are formed. The system then processes the raw image from each channel to identify cells, and then determines absolute cell counts and relative antigen density levels for each type of cell labeled with a fluorescent antibody.
Marker MLSC can also be used to quantitate soluble factors in biological fluids by using a microsphere-bound primary antibody to the factor along with a secondary fluorescently-labeled antibody to the factor. The factor thereby becomes bound to the microsphere, and the binding of the secondary antibody fluorescently labels the bound factor. The system in this embodiment measures the fluorescent signal associated with each bead in the blood sample in order to determine the concentration of each soluble factor. It is possible to perform multiple assays in the same sample volume by using multiple bead types (each conjugated to a different primary antibody). In order to identify each bead type, the different beads can have distinct sizes or can have a different internal color, or each secondary antibody can be labeled with a different fluorophore.
Although preferred embodiments of the invention use antibodies to detect biological markers, any other detection molecule capable of binding specifically to a particular biological marker is contemplated. For example, various types of receptor molecules can be detected through their interaction with a fluorescently-labeled cognate ligand.
The raw data from the MLSC instrument is processed by image analysis software to produce data about the cell populations and soluble factors that were the subject of the assay. This data is then transferred to a database. Other data that can be stored along with this cell population and soluble factor data for the purposes of establishing correlations between biological markers and diseases or medical conditions include: drug dosing and pharmacokinetics (measurement of the concentrations of a drug and its metabolites in a body); clinical parameters including, but not limited to, the individual's age, gender, weight, height, body type, medical history (including co-morbidities, medication, etc.), manifestations and categorization of disease or medical condition (if any) and other standard clinical observations made by a physician. Also included among the clinical parameters would be environmental and family history factors, as well as results from other techniques for measuring the concentrations of specific molecules present in the bodily fluids of the individual, including, without limitation, standard ELISA tests, colorimetric functional assays for enzyme activity, and mass spectrometry. Data may also include images such as x-ray photographs, brain scans, or MRIs, or information obtained from biopsies, EKGs, stress tests or any other measurement of an individual's condition.
An informatics system then a) compares the data with stored profiles (either from the same individual for disease progression or therapeutic evaluation purposes and/or from other individuals for disease diagnosis); and b) “mines” the data in order to derive new profiles. In this way, diagnostic and prognostic information can be obtained from and derived by the database. U.S. Provisional Patent Application Serial No. 60/131,105, filed Apr. 26, 1999, entitled “Biological Marker Identification System,” and the commonly-owned United States Utility Application filed concurrently with this application, entitled “Phenotype and Biological Marker Identification System,” each of which is specifically incorporated herein by reference in its entirety, describes in great detail the use of MLSC in many different applications. The system is capable of providing robust and consistent assay data, even in assays in which prior art systems are hindered by variability among donor samples. Applications include the use of MLSC to measure cell-type population changes and soluble factor changes during disease progression and during therapy. For example, MLSC may be used to identify novel biological markers for multiple sclerosis and rheumatoid arthritis.